Nanopore-based analysis of analytes

Abstract

The present disclosure relates to systems and methods for analysis of proteins, more in particular to nanopore systems, devices and methods for single-molecule protein analysis and sequencing. Provided is a method for translocating a target protein through a nanopore, the nanopore being comprised in a membrane separating a fluidic chamber of a nanopore system into a cis side and a trans side, comprising:(a) allowing a protein translocase in solution to capture and form a complex with the target protein to be translocated;(b) contacting the translocase-target protein complex with the cis side of the nanopore and allowing for translocation of the target protein to the trans side; wherein the nanopore system has a cis to trans electro-osmotic force (EOF) resulting from a large net ionic current flow cis-to-trans relative to the total ionic current flow, so that the target protein is captured in the nanopore with on top of the nanopore the translocase controlling the translocation.

What is claimed is: 
     
         1 .- 20 . (canceled) 
     
     
         21 . A method comprising:
 (a) providing:
 (i) a nanopore system, wherein the nanopore system comprises (1) a fluidic chamber, (2) a membrane that separates the fluidic chamber into a first side and a second side, and (3) at least a portion of a nanopore disposed in the membrane; 
 (ii) a non-nucleic acid based polymer analyte; and 
 (iii) a translocase; 
   (b) translocating at least a first portion of the non-nucleic acid based polymer analyte from the first side toward the second side; and   (c) translocating at least a second portion of the non-nucleic acid based polymer analyte toward the first side.   
     
     
         22 . The method of  claim 21 , wherein the at least the first portion of the non-nucleic acid based polymer analyte and the at least the second portion of the non-nucleic acid based polymer analyte are the same portion of the non-nucleic acid based polymer analyte. 
     
     
         23 . The method of  claim 21 , wherein the non-nucleic acid based polymer analyte comprises a peptide, a polypeptide, a protein, or any combination thereof. 
     
     
         24 . The method of  claim 21 , wherein the non-nucleic acid based polymer analyte is coupled to at least one leader construct. 
     
     
         25 . The method of  claim 24 , wherein the leader construct comprises a stall motif, a block motif, a coupling motif, or any combination thereof. 
     
     
         26 . The method of  claim 21 , further comprising contacting the non-nucleic acid based polymer analyte with the translocase. 
     
     
         27 . The method of  claim 26 , wherein the contacting occurs outside of the nanopore system. 
     
     
         28 . The method of  claim 21 , wherein the translocase is configured to disrupt a quaternary, tertiary, or secondary structure of the non-nucleic acid based polymer analyte. 
     
     
         29 . The method of  claim 21 , wherein the translocase is an adenosine triphosphate (ATP)-driven unfoldase or a nucleotide triphosphate (NTP)-driven unfoldase. 
     
     
         30 . The method of  claim 21 , wherein the translocase is a protein translocase. 
     
     
         31 . The method of  claim 21 , wherein the translocating of (b) is performed using an electrophoretic force (EPF). 
     
     
         32 . The method of  claim 21 , wherein the translocating of (b) is performed using an electro-osmotic force (EOF). 
     
     
         33 . The method of  claim 21 , wherein the translocating of (c) is performed in part by the translocase. 
     
     
         34 . The method of  claim 33 , wherein the translocase is moving in an opposite direction as compared to a movement of the at least the second portion of the non-nucleic acid based polymer analyte. 
     
     
         35 . The method of  claim 21 , further comprising measuring a signal generated by the translocating of (b), the translocating of (c), or a combination thereof. 
     
     
         36 . The method of  claim 35 , wherein the measuring comprises measuring the signal for states of (i) an open channel of the nanopore; (ii) capture of the at least the first portion of the non-nucleic acid based polymer analyte or the at least the second portion of the non-nucleic acid based polymer analyte by the nanopore; or (iii) passage of the at least the first portion of the non-nucleic acid based polymer analyte or the at least the second portion of the non-nucleic acid based polymer analyte in the nanopore. 
     
     
         37 . The method of  claim 21 , wherein the first portion of the non-nucleic acid based polymer analyte is translocated from the first side toward the second side or the second portion of the non-nucleic acid based polymer analyte is translocated toward the first side. 
     
     
         38 . The method of  claim 21 , wherein the nanopore comprises an inner pore constriction from about 0.2 nanometers (nm) to about 10 nm. 
     
     
         39 . The method of  claim 21 , wherein the nanopore is an alpha-helical pore forming toxin or porin, or a beta-barrel oligomeric pore forming toxin or porin. 
     
     
         40 . The method of  claim 21 , wherein the non-nucleic acid based polymer analyte is coupled to a nucleic acid molecule.